Synaptic and nonsynaptic localization of protocadherin‐γC5 in the rat brain

Abstract

It has been proposed that γ-protocadherins (Pcdh-γs) are involved in the establishment of specific patterns of neuronal connectivity. Contrary to the other Pcdh-γs, which are expressed in the embryo, Pcdh-γC5 is expressed postnatally in the brain, coinciding with the peak of synaptogenesis. We have developed an antibody specific for Pcdh-γC5 to study the expression and localization of Pcdh-γC5 in brain. Pcdh-γC5 is highly expressed in the olfactory bulb, corpus striatum, dentate gyrus, CA1 region of the hippocampus, layers I and II of the cerebral cortex, the molecular layer of the cerebellum. Pcdh-γC5 is expressed in both neurons and astrocytes. In hippocampal neuronal cultures, and in the absence of astrocytes, a significant percentage of synapses, more GABAergic than glutamatergic, have associated Pcdh-γC5 clusters. Some GABAergic axons show Pcdh-γC5 in the majority of their synapses. Nevertheless, many Pcdh-γC5 clusters are not associated with synapses. In the brain, a significant number of Pcdh-γC5 clusters are located at contact points between neurons and astrocytes. Electron microscope immunocytochemistry of the rat brain shows that i) Pcdh-γC5 is present in some GABAergic and glutamatergic synapses both pre- and postsynaptically; ii) Pcdh-γC5 is also extrasynaptically localized in membranes and in cytoplasmic organelles of neurons and astrocytes; and iii) that Pcdh-γC5 is also localized in perisynaptic astrocyte processes. The results support the notion that i) Pcdh-γC5 plays a role in synaptic specificity and/or synaptic maturation, and ii) that Pcdh-γC5 is involved in neuron-neuron synaptic interactions and in neuron-astrocyte interactions, including perisynaptic neuron-astrocyte interactions.

Morphological plasticity of astroglia: Understanding synaptic microenvironment

“…These mechanisms have been implicated when observing patients with autism, a disease linked to mutations in neuroligins and neurexins (Bernardinelli et al, 2014a; Chih et al, 2005). Other adhesive molecules that have been involved in neuron‐astrocyte binding and that might be involved in boosting PAP morphogenesis are Ng‐CAM (Grumet et al, 1984), SynCAM1 (Sandau et al, 2012), NCAM (Theodosis et al, 2004), and protocadherin‐gammaC5 (Garrett and Weiner, 2009; Li et al, 2010). However, whether and how these signaling molecules and their partners or ligands act within PAPs in organized tissue in situ remains to be established.…”

Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit

“…Preparation of Rat Forebrain Homogenates and Subcellular Fractionation-The preparation of rat forebrain homogenates and various subcellular fractions, including crude synaptosomal (P2), synaptosomal (P2B), microsomal (P3), synaptic plasma membrane (SPM), and "one Triton" postsynaptic den-sity (PSD) fractions, has been described elsewhere (35,37). For membrane preparation, the procedure has been described previously (37).…”

“…For membrane preparation, the procedure has been described previously (37). Briefly, the forebrain (telencephalon) tissue of adult female Sprague-Dawley rats was homogenized with 10% sucrose in 50 mM Tris-HCl, pH 7.4 buffer, and centrifuged for 5 min at 1,000 ϫ g. The supernatant was centrifuged at 100,000 ϫ g for 1 h, and the pellet was suspended in 5 mM Tris-HCl, pH 7.4 buffer, and then subjected to homogenization in an all-glass Dounce homogenizer.…”

“…Immunofluorescence of Rat Brain Sections-The procedure has been described elsewhere (36,37). Briefly, 25-m-thick brain slices (prepared as described above) from a P90 SpragueDawley rat were subjected to an antigen retrieval procedure by incubation with pepsin (0.15 mg/ml in 0.2 N HCl) at 37°C for 5 min to facilitate the exposure of the tissue antigens to the antibodies.…”

“…Peter Somogyi and Zoltan Nusser, and the preparation of the blocks was described elsewhere (49). The tissue blocks were sectioned in our laboratory, and the ultrathin sections were subjected to the immunogold labeling procedure as described elsewhere (28,33,36,37,39). The tissue sections were incubated first with a mixture of affinity-purified Rb anti-RNF34 and Ms mAb to ␤2/3 GABA A R subunits, followed by incubation with goat anti-rabbit and goat anti-mouse IgG secondary antibodies labeled with colloidal gold particles of 18 and 10 nm in diameter, respectively.…”